Genome-Wide Assessment of Stress-Associated Genes in Bifidobacteria

ABSTRACT Over the last decade, the genomes of several Bifidobacterium strains have been sequenced, delivering valuable insights into their genetic makeup. However, bifidobacterial genomes have not yet been systematically mined for genes associated with stress response functions and their regulation. In this work, a list of 76 genes related to stress response in bifidobacteria was compiled from previous studies. The prevalence of the genes was evaluated among the genome sequences of 171 Bifidobacterium strains. Although genes of the protein quality control and DNA repair systems appeared to be highly conserved, genome-wide in silico screening for consensus sequences of putative regulators suggested that the regulation of these systems differs among phylogenetic groups. Homologs of multiple oxidative stress-associated genes are shared across species, albeit at low sequence similarity. Bee isolates were confirmed to harbor unique genetic features linked to oxygen tolerance. Moreover, most studied Bifidobacterium adolescentis and all Bifidobacterium angulatum strains lacked a set of reactive oxygen species-detoxifying enzymes, which might explain their high sensitivity to oxygen. Furthermore, the presence of some putative transcriptional regulators of stress responses was found to vary across species and strains, indicating that different regulation strategies of stress-associated gene transcription contribute to the diverse stress tolerance. The presented stress response gene profiles of Bifidobacterium strains provide a valuable knowledge base for guiding future studies by enabling hypothesis generation and the identification of key genes for further analyses. IMPORTANCE Bifidobacteria are Gram-positive bacteria that naturally inhabit diverse ecological niches, including the gastrointestinal tract of humans and animals. Strains of the genus Bifidobacterium are widely used as probiotics, since they have been associated with health benefits. In the course of their production and administration, probiotic bifidobacteria are exposed to several stressors that can challenge their survival. The stress tolerance of probiotic bifidobacteria is, therefore, an important selection criterion for their commercial application, since strains must maintain their viability to exert their beneficial health effects. As the ability to cope with stressors varies among Bifidobacterium strains, comprehensive understanding of the underlying stress physiology is required for enabling knowledge-driven strain selection and optimization of industrial-scale production processes.

Over the past 2 decades, the morphological, physiological, and metabolic stress responses of bifidobacteria have been increasingly studied to understand the molecular mechanisms that underlie their stability and robustness . The stress responses of Bifidobacterium strains with diverse stability properties have been assessed, applying traditional methodologies (2-7, 14, 25, 27-32) and, more recently, omics technologies (8-13, 15-24, 26) to characterize and compare strains. The response and tolerance of bifidobacteria to a particular stressor was found to differ significantly among species but also among strains (33,34). This diversity hampers the extrapolation of data from well-studied strains, such as B. longum subsp. longum NCC2705 and B. breve UCC2003, to even closely related strains. However, understanding the molecular mechanisms underlying stability and robustness in bifidobacteria is crucial for driving rational strain selection as well as knowledge-driven optimization of production processes.
Variation in stress tolerance across Bifidobacterium strains might be explained by their genetic diversity in terms of stress-associated genes. In 2002, Bifidobacterium longum subsp. longum NCC2705 was the first Bifidobacterium strain to have its genome sequenced (35). In the following years, multiple genomes of Bifidobacterium strains have been sequenced, resulting in more than 150 complete genome sequences and even more draft sequences being publicly available. Previous comparative analyses of bifidobacterial genomes have mainly focused on the evolution of the genome and niche-specific adaptations, paying special attention to their carbohydrate utilization capabilities (36)(37)(38)(39)(40)(41)(42)(43)(44). Some genomic studies have focused on the entire genus (36)(37)(38)(39)(40)(41)(42), whereas others have targeted individual species (43,44). A number of studies highlighted the high genomic diversity across Bifidobacterium species (38,39), but to date only a single study has touched upon the prevalence of stress-associated genes in bifidobacteria (42).
The aim of the present study was to explore the prevalence of known stress-associated genes among bifidobacteria with publicly available genome sequences to gain insights into the diversity of stress physiology in this important group of bacteria. The obtained stress response gene profiles were combined with the current knowledge on stress tolerance of Bifidobacterium strains to identify genotype-phenotype correlations. In addition, previous findings on the stress response mechanisms were assessed, in terms of their genus-wide validity, and novel hypotheses were generated.

RESULTS AND DISCUSSION
In this study, the prevalence of stress-associated genes in bifidobacterial genomes was investigated to gain new insight into the diverse stress physiology of the genus. A list of 76 genes that have been previously implicated in stress responses of Bifidobacterium strains was compiled through an extensive literature survey. The genes were grouped into six categories: (i) protein quality control (PQC) and DNA repair systems, (ii) oxidative stress, (iii) acid stress, (iv) bile stress, (v) organic solvent stress, and (vi) putative transcriptional regulators of stress response. With the single exception of the glutamate/gamma-aminobutyrate antiporter GadC, genes encoding transporters were not included in the study due to the difficulty of computationally determining the substrate specificity based solely on their protein sequence. All stress-associated genes included in this study are listed in Table S1 in the supplemental material.
The distribution of the selected genes was assessed in 171 Bifidobacterium strains (Table S2), representing 22 species (Table 1), through protein homology search. In total, 9,362 hits were identified. The complete list of homologs is provided in Data Set S1.
Clear differences in the presence of stress-associated genes were detected among previously suggested phylogenetic groups of bifidobacteria (37,40) and among species (Fig.  1). In addition, a few differences were observed among strains of the same species (Fig.  S1). Slightly fewer than half of the studied stress-associated genes were found in all studied strains or missing in strains of only 1 out of the 22 species (i.e., present in 95% of the species), whereas 53% of the studied stress-associated genes were identified in fewer than 95% of the species (Fig. 2A). Homologs of two studied genes (3%) were only identified in B. subtile KCTC 3272 ( Fig. 2A), which was the only representative of the B. subtile species. The analysis showed that most genes associated with the PQC and DNA repair systems, i.e., 64%, are well conserved across species, whereas more than half of the genes related to oxidative stress, acid stress, and putative transcriptional regulators of stress responses, i.e., 62%, 78%, and 75%, respectively, were present in fewer than 95% of species (Fig. 2B). The degree of sequence similarity of the stress-associated genes across species was highly diverse (Fig. 1). Cutoffs of 40% sequence identity and 70% coverage were applied for the homology search to reduce the risk of including homologs with function dissimilar to that of the query protein. Most of the identified homologs shared at least 50% sequence identity ( Fig. 1), and the alignment covered more than 80% of the query stress protein sequence (Data Set S1). However, for some stress-associated genes the sequence similarity was lower across some homologs, particularly for query protein sequences or hits from the genomes of strains that belongs to the B. asteroides group.
To investigate the correlation between the presence of stress-associated genes and actual stress tolerance of Bifidobacterium strains, data on the tolerance to individual stressors was collected (see Table S3). In the following sections, each category of stress-associated genes will be discussed along with the current knowledge on the studied genes and the homologs identified in this study. When very low protein sequence similarity was detected across homologs, the sequences of the homologs were more thoroughly compared through multiple-sequence alignment.
Protein quality control and DNA repair systems. Different stresses, such as oxidative and heat stress, have a detrimental effect on macromolecules, including DNA and proteins, making the PQC system and DNA repair systems crucial for a cell's stress resistance (45,46). So-called heat shock proteins (Hsps), which function as chaperones, cochaperones, and proteases, are major constituents of the PQC system in bacteria (46). Despite their naming, they are also involved in responses to stresses other than heat (46). (i) Most genes of the protein quality control and DNA repair systems are highly conserved. Most information available on the genomic organization and regulation of Hsps in bifidobacteria derives from extensive studies on B. breve UCC2003 exposed to heat, osmotic, and organic solvent stress (24,47). Based on genetic and transcriptional studies, a model for the stress gene regulatory network, covering the regulation of the PQC system, has been proposed (24). The model includes 11 Hsps (ClpC, ClpP1, ClpP2, GroEL, GroES, ClpB, ClpX, DnaK, GrpE, DnaJ, and DnaJ2) and three transcriptional regulators (the repressors HspR and HrcA and the activator ClgR) (24). The transcriptional regulation of Hsps has been proposed to overlap those of the DNA repair system (24). Based on the high degree of conservation of the regulons and binding sites, the model of the stress gene regulatory network in B. breve UCC2003 was suggested to be valid for all Bifidobacterium species characterized (24). Moreover, the presence of cold shock protein A (CspA), encoded next to GroEL (31), and universal stress protein A (UspA), encoded next to ClpC in B. breve UCC2003, was suggested to be widely conserved in bifidobacteria (6). Since the availability of bifidobacterial genomes was still rather limited at the time of these studies, we assessed the general validity of the proposed model using the genome sequences available today.  (37,40). For each stressassociated gene, a query protein sequence was extracted from the genome of a strain in which it was proposed to be involved in stress responses. Homologs of stress-associated gene products across the 22 species were identified using DIAMOND BLASTp (E value, 0.001; sequence identity cutoff, 40%; coverage cutoff, 70%). The maximum likelihood phylogeny tree was constructed using CLC Genomics Workbench. The tree scale gives the average number of substitutions per site. Information on the stress-associated gene products can be found in Table S1.
Searching all 171 bifidobacterial genomes for orthologs of the genes encoding the described PQC and DNA repair systems in B. breve UCC2003 confirmed that, with very few exceptions, all analyzed Bifidobacterium strains possess all these genes ( Fig. 1 and Fig. S1). However, the degree of sequence conservation varied widely with the different genes ( Fig.  1), suggesting that the regulatory network is of fundamental importance in bifidobacteria but might have some unknown functional variations across species.
In contrast to previous findings that the cspA-groEL organization and uspA-clpC organization are widely conserved in bifidobacteria (6,31), our genomic analysis revealed that CspA and UspA were present in less than 95% of the bifidobacterial genomes. A homolog of UspA was not detected in strains isolated from the digestive tract of bees (B. asteroides group) in B. pullorum subsp. gallinarum CACC 514 (only representative of B. pullorum species) ( Fig. 1) or in some strains of B. breve (Fig. S1). Moreover, all strains belonging to the B. adolescentis group, B. angulatum, B. indicum LMG 11587 (only representative of B. indicum species), and B. pullorum subsp. gallinarum CACC 514 (Fig. 1), and some strains of B. breve lacked a homolog of CspA (Fig. S1). The lack of sufficient knowledge on the function of CspA and UspA in bifidobacteria makes the biological implication of the absence of these genes ambiguous. In general, cold shock proteins might serve as RNA chaperones under stress conditions (48), and universal stress proteins might have a role in DNA protection (49). In B. breve UCC2003, the cspA gene was induced upon heat stress (24,31), whereas the uspA gene was not (6). Interestingly, a second universal stress protein gene (UspA_2), upregulated upon bile stress in B. breve UCC2003 (19), and a gene encoding a cold shock domain-containing protein (CspB), located on the gene segment between groEL and clpC, were found to be conserved in all strains of all studied species (Fig. 1). Although the role of CspB remains to be elucidated, it seems not to be involved in the heat stress response in bifidobacteria (50).
Some proposed characteristics of the bifidobacterial heat shock system are uncommon for high-G1C Gram-positive bacteria (6,31). High-G1C Gram-positive bacteria, such as members of the genus Streptomyces, contain several copies of clpC (51) and groEL (52), whereas bifidobacterial genomes were suggested to contain only a single copy of these genes (6,31). This was confirmed for all genomes included in this study. Furthermore, additional members of the Hsp100 family, including ClpA, ClpE, and ClpY as well as heat shock proteins of the Hsp33 and Hsp90 families, were confirmed to be absent from all sequenced bifidobacterial genomes based on functional annotation information in the NCBI Reference Sequence (RefSeq) database (47).
(ii) The regulation of the protein quality control and DNA repair systems may vary across phylogenetic groups. The regulatory network of the PQC and DNA repair systems in B. breve UCC2003 was suggested to be under the control of the transcriptional repressors HspR (heat shock protein repressor) and HrcA (heat regulation at controlling inverted repeat of chaperone expression [CIRCE]) as well as the transcriptional activator ClgR (Clp gene regulator) (24). The proposed consensus motifs of the regulators and their position in the genome of B. breve UCC2003 are shown in Table 2. The model proposed for the regulation of the PQC and DNA repair systems in bifidobacteria is highly based on these binding motifs and their position in the genome of B. breve UCC2003. Thus, after confirming the high degree of conservation of the included genes, the general validity of the proposed model was further reviewed by assessing the conservation of the binding motifs of the regulators across different strains of the genus. For this purpose, the genome sequences B. longum subsp. longum NCC2705, belonging to the same phylogenetic group as B. breve UCC2003, and B. animalis subsp. lactis BB-12 and B. adolescentis ATCC 15703, representing two other phylogenetic groups ( Fig. 1) with different stability characteristics (Table S3), were screened for the three motifs using Find Individual Motif Occurrences (FIMO) (53).
The gene organization of the PQC and DNA repair systems was confirmed to be identical to that in B. breve UCC2003 in all three strains, except for the absence of a CspA homolog in B. adolescentis ATCC 15703. As in B. breve UCC2003, an HspR-associated inverted repeat (HAIR) motif was detected twice upstream of the dnaK operon in B. longum subsp. longum NCC2705 and B. adolescentis ATCC 15703 (Table 2). In contrast, in B. animalis subsp. lactis BB-12, the motif appears only once in the promoter region of dnaK and shows slight deviations from the suggested consensus sequence (ATACTTGAGTGA-N 6 -CTCAAGTTTT), with a tyrosine inserted into the adenine-rich extension. The presence of a single HAIR motif upstream of the dnaK operon was described for B. animalis subsp. lactis DSM 10140 (4), suggesting that this is a specieswide characteristic. No HAIR motif could be detected upstream of the clgR gene in B. animalis subsp. lactis BB-12 and B. adolescentis ATCC 15703 (Table 2), whereas in B. longum subsp. longum NCC2705 the HAIR motif upstream of the clgR gene lies inside the coding region of a gene predicted to encode a CinA family protein. Genome-wide in silico screening for the motif further revealed that, unlike the other strains, B. animalis a Consensus sequences of the motifs have been previously proposed by Zomer et al. (24). The nucleotide codes of the motifs follow the IUPAC nomenclature. b HAIR, HspR-associated inverted repeats. The number of Ns in the HAIR sequence was incorrectly given as five in the original publication. c CIRCE, controlling inverted repeat of chaperone expression sequence. subsp. lactis BB-12 only has one CIRCE-like motif upstream of the groES gene (Table 2). Several nonsignificant hits for the ClgR-like motif were detected in B. longum subsp. longum NCC2705, B. animalis subsp. lactis BB-12, and B. adolescentis ATCC 15703. Therefore, the sequences upstream of the potentially ClgR-regulated genes were screened for the ClgR motif in individual analyses. A ClgR-like motif was detected in the promoter region of the clpP operon, upstream of hrcA and clpC in B. longum subsp. longum NCC2705 (hit upstream of HrcA has a q-value above 0.01), but not in B. animalis subsp. lactis BB-12. In B. adolescentis ATCC 15703, the ClgR motif was only detected upstream of the clpC gene and the clpP operon (Table 2).
Taken together, these findings suggest that the regulation of the PQC and DNA repair systems in B. animalis subsp. lactis BB-12 and B. adolescentis ATCC 15703 differs from the regulation in B. breve UCC2003 and B. longum subsp. longum NCC2705. In particular, the function of the activator ClgR in B. animalis subsp. lactis BB-12 and B. adolescentis remains obscure. Consequently, the proposed model of the stress regulatory network might be valid for rather closely related strains of B. breve UCC2003 but not for all strains of the genus. Thus, the regulation of the PQC and DNA repair systems merits further research, with a focus on strains outside the B. longum group.
(iii) Multiple species lack an Hsp20 homolog. Besides the already-described genes, the serine protease HtrA and the small heat shock protein Hsp20 were suggested to be part of the stress-induced PQC system. Both bile and severe heat stress were observed to induce the expression of htrA in Bifidobacterium strains (21,22,50,54). Homologs of HtrA were found to be present in all analyzed genomes except three B. longum strains (Fig. S1). Hsp20 was found to be strongly induced upon multiple stresses, including heat, high osmolality, hydrogen peroxide (H 2 O 2 ), and starvation, and was proposed as a suitable biomarker for stress (13,30). The analysis of this study showed that only 73% of the Bifidobacterium strains possess genes encoding homologs of Hsp20 from B. breve UCC2003. The presence of an Hsp20 homolog seems to be species dependent, except for that in B. longum strains (Fig. S1). In contrast to the previous hypothesis that Hsp20 is exclusive for isolates from the human intestine (30), an Hsp20 homolog was also identified in isolates from bees, such as B. asteroides PRL2011 (51% identity, 99% coverage) ( Fig. 1 and Data Set S1). However, no Hsp20 homolog was found in other animal isolates (Fig. 1), including B. animalis subsp. lactis BB-12. Furthermore, the analysis contradicts previous results of a slot plot hybridization test, demonstrating the presence of an hsp20 gene in the human isolate B. catenulatum DSM 16992 (LMG 11043) (30); no gene encoding Hsp20 was identified in any B. catenulatum strain, including DSM 16992 (Fig. 1). Since animal isolates, including B. animalis subsp. lactis BB-12, possess high heat tolerance (33), it can be excluded that Hsp20 is essential for sufficient protection of the cells against heat shock. Rather, Hsp20 may be important for coping with stressors other than heat, as suggested before (30). In other organisms, small heat shock proteins, such as Hsp20, have been linked to functions other than classical chaperones, e.g., biofilm formation and cell protection during dormancy (55). Interestingly, animal isolates have been shown to commonly grow at higher temperatures than human isolates (up to 45°C) (56). Therefore, it is conceivable that high expression of Hsp20 upon heat stress is linked to a slow down of growth in bifidobacteria, as has been observed for HspX in Mycobacterium tuberculosis (57). To increase the understanding of the relationship between the presence of Hsp20 and the stress resistance of Bifidobacterium strains, its biological function should be further investigated, e.g., by studying its transcriptional regulation (30) as well as the effect of knocking out the gene on growth and survival at elevated temperatures.
Oxidative stress response. As for other stressors, the sensitivity to oxidative stress varies significantly among Bifidobacterium strains, but the molecular basis of this variation is not fully understood (33,58). The toxicity of O 2 is mainly linked to the formation of so-called reactive oxygen species (ROS), including H 2 O 2 , superoxide anions (O 2 Á2 ), and hydroxyl radicals (OH Á ). In contrast to aerobic bacteria, O 2 -sensitive bacteria lack an efficient system to detoxify ROS. For the elimination of oxidative stress, the interplay of O 2 -scavenging and ROS-detoxifying enzymes is critical to avoid accumulation of H 2 O 2 , which is produced by some of these enzymes and decomposed by others.
In line with the different O 2 tolerances of Bifidobacterium strains, most of the 34 investigated genes that are related to oxidative stress were not conserved across species (Fig. 1). In addition, some oxidative stress-associated genes had very low protein sequence similarity across species (Fig. 1), as discussed further below.
(i) Bee-specific strains harbor unique genetic characteristics linked to oxygen tolerance. Isolates from the digestive tract of bees have been shown to have exceptionally high O 2 tolerance (Table S3) (59), which might derive from adaptation to elevated O 2 concentrations in their natural environment (60). Previous genome analysis of the aerotolerant B. asteroides PRL2011 revealed genes encoding enzymes of an electron transport chain for aerobic respiration (59), which had also been detected in additional bee isolates (39). In agreement, we identified homologs for a H 1 -translocating NADH dehydrogenase, cytochrome d oxidase (CydA, CydB, CydC, and CydD), succinate dehydrogenase subunits (SdhA and SdhB), and all subunits of F 1 F 0 -ATPase in all bee isolates included in the analysis. Homologs of all these proteins were further found in B. subtile KCTC 3272 ( Fig. 1), an isolate from sewage, the living host of which remains unknown (61).
Despite the presence of genes encoding an electron transport chain in all four strains, O 2 consumption was previously only detected in B. asteroides PRL2011 but not in B. coryneforme LMG 18911, B. indicum LMG 11587, and B. actinocoloniiforme DSM 22766 (59). The absence of other respiratory-associated genes in these strains, such as the hydrogen peroxide-dependent heme synthase (based on functional annotation information in the NCBI database), may explain the observed phenotypical difference.
Homologs of the succinate dehydrogenase subunits SdhA and SdhB were detected in all studied genomes (Fig. 1), suggesting a housekeeping function (59). As all strains lacked homologs of the heme-binding membrane domain subunit (SdhC and SdhD) of the enzyme, it appears to exist as a soluble enzyme. Low sequence similarity (42 to 67%) was detected between the genes encoding SdhA and SdhB from B. asteroides PRL2011 (query) and their homologs in other species (Fig. 1). In nonrespiring strains, the enzyme might rather function as fumarate reductase, catalyzing the opposite reaction of succinate dehydrogenase (62).
The F 1 F 0 -ATPase (ATP synthase) was highly conserved across genomes of the investigated species (Fig. 1). In general, F 1 F 0 -ATPase can catalyze the synthesis of ATP using the energy of an electrochemical ion gradient or convey the extrusion of protons under low driving force conditions, such as acid stress, using ATP for energy supply (63). In nonrespiring strains, the enzyme is thought to be crucial for the maintenance of a proton gradient (64), whereas in respiring strains, such as B. asteroides PRL2011, it might contribute to ATP synthesis as part of the electron transport chain (59). The sequences of the alpha-and beta-subunits, forming the catalytic site of the enzyme in the F 1 domain, were found to be highly conserved across strains in our analysis. In contrast, relatively low sequence similarity was detected for other subunits of the F 1 F 0 -ATPase, particularly for the epsilon subunit (part of the rotor) and the delta subunit (part of the stator) of F 1 F 0 -ATPase (Fig. 1).
Most Bifidobacterium strains are superoxide dismutase (SOD) negative and catalase negative. However, genes predicted to encode SOD and catalase have been identified in the bee isolates, e.g., B. xylocopae subsp. nov. XV2 and B. asteroides PRL2011 (59,65), and an oxygen-inducible heme catalase has been characterized in B. asteroides DSM 20089 (66). Our genomic analysis revealed that all strains that harbor genes for a putative electron transport chain also harbor homologs of SOD from B. xylocopae XV2 (Fig. 1), whereas only B. asteroides and B. actinocoloniiforme DSM 22766 (the only representative of B. actinocoloniiforme species) were found to have a catalase homolog (Fig. 1). The SOD and catalase genes in these strains showed high sequence identity (91% at 100% coverage) to SOD genes from the bee isolate Bombiscardovia coagulans (67).
Even though homologs of SOD from B. xylocopae XV2 were only detected in bee isolates, previous studies have reported SOD activity, albeit very low, in Bifidobacterium strains isolated from other sources (68)(69)(70). Spontaneous destruction of O 2 Á2 might explain low SOD activity in strains lacking an SOD homolog (71). Nonetheless, we searched bifidobacterial genomes in NCBI GenBank for the presence of genes annotated as SOD aside from the SOD homologs from B. xylocopae XV2 and detected a gene predicted to encode an SOD in B. longum subsp. infantis ATCC 15697 (Blon_1406, WP_003829426.1). This strain has been reported to have low SOD activity and to be hypersensitive to O 2 (28,69,70). The putative SOD gene from B. longum subsp. infantis ATCC 15697 is significantly shorter than the SOD homologs in bee isolates (132 versus 206 amino acids). The putative SOD homolog was included in our homology search. Homologs with high sequence similarity to the putative SOD gene were detected in a subset of B. breve strains and B. longum subsp. infantis strains as well as in four bee isolates, in the latter with only 50% protein sequence identity (Fig. S1). It remains to be confirmed if the gene actually encodes an SOD that might negatively influence the O 2 tolerance in strains lacking a sufficient H 2 O 2 detoxification system.
(ii) Oxidative stress-associated genes from B. tibiigranuli are rare across species. Genomic analysis of the water kefir isolate B. tibiigranuli TMW 2.2057 identified genes, including pyruvate oxidase, glutathione peroxidase, and the peroxide stress protein YaaA, that potentially contribute to O 2 tolerance (18). While homologs of all three gene products were found in B. subtile KCTC 3272, no homologs for glutathione peroxidase and YaaA were found in strains of another species (Fig. 1), and only homologs with very low similarity to pyruvate oxidase (40 to 41%) were identified in some bee isolates (Fig. 1). These results confirm the previous findings that B. tibiigranuli strains have several unique genetic traits among bifidobacteria and that the species is phylogenetically close to B. subtile (18).
(iii) The bifidobacterial HemN might function as HemW. Under aerobic conditions, the expression of a gene predicted to encode an oxygen-independent coproporphyrinogen III oxidase (HemN) was induced in the O 2 -tolerant B. animalis subsp. lactis IPLA4549 and an ahpC-overexpressing mutant of B. longum subsp. longum NCC2705 (20,27). Our genomic analysis detected homologs of HemN from B. longum subsp. longum NCC2705 in all analyzed strains, with lowest sequence similarity (55 to 59%) to the reference in members of the B. pseudolongum and B. asteroides group (Fig. 1).
The role of HemN (a key enzyme in heme biosynthesis) in the oxidative stress response remains to be understood (20). In contrast to its annotation in the literature, the HemN homolog from multiple strains was annotated as radical SAM family heme chaperone HemW based on the Prokaryotic Genome Annotation Pipeline (PGAP) functional annotation (Table  S1). This held true for HemN homologs from B. longum subsp. longum NCC2705 as well as from 33 additional B. longum strains, B. coryneforme LMG18911, and B. indicum LMG 11587 (Data Set S1). In line with this, in Lactococcus lactis, a protein originally annotated as HemN was previously found to lack coproporphyrinogen III oxidase activity and was instead assigned to the protein HemW family with a putative function as a heme chaperone (72).
Due to the inconclusive annotation of the homologs in Bifidobacterium strains, its protein sequence in B. longum subsp. longum NCC2705, B. animalis subsp. lactis BB-12, and B. asteroides PRL2011 was scanned for conserved amino acid motifs specific to the HemW family to investigate if HemN in bifidobacteria also belongs to the HemW family. All HemW-specific motifs were detected in the gene products of the three strains (Fig. 3), suggesting that also in bifidobacteria, the enzyme does not function as HemN but rather shares its function with HemW in L. lactis.
However, L. lactis strains can respire in the presence of exogenous hemin, whereas most Bifidobacterium strains lack enzymes required for respiratory growth, which means that the function of HemW in bifidobacteria appears superfluous. Moreover, coproporphyrinogen III oxidase activity has been detected in cell extract of B. animalis subsp. lactis IPLA 4549 (20). Assessing the heme-binding affinity of the bifidobacterial HemN/W as well as the effect of heme addition on its expression and on the O 2 tolerance of strains might help to understand the role of HemN/W in bifidobacteria.
(iv) H 2 O 2 -forming NAD(P)H oxidases are encoded in both oxygen-tolerant and -sensitive species. In many studies, low oxidative stress tolerance in bifidobacteria has been linked to H 2 O 2 formation and its insufficient detoxification under aerobic conditions (69,70,73,74). To date, two H 2 O 2 -forming NAD(P)H oxidases have been characterized in Bifidobacterium strains: a b-type dihydroorotate dehydrogenase (DHOD) from the O 2 -sensitive B. bifidum ATCC 29521 and an NADPH oxidase (NPOX) from the O 2 -hypersensitive B. longum subsp. infantis ATCC 15697 (28,29). Even though these enzymes have been suggested to contribute to O 2 sensitivity (28, 29), homologs of DHOD (PyrK and PyrDb subunit) and NPOX were also identified in the genomes of O 2 -tolerant strains, such as B. animalis subsp. lactis BB-12 (Fig. S1). Overall, 94% and 89% of the analyzed strains possessed PyrK, PyrDb, and NPOX homologs (Fig. S1). Therefore, it can be concluded that the sole presence of these genes is not indicative of O 2 sensitivity. It seems more likely that the expression level of H 2 O 2 -forming NAD(P)H oxidases (28) and, in their presence, the activity of H 2 O 2 -detoxifying enzymes determine a strain's ability to cope with O 2 . In line with this, a previous study reported that heterologous expression of npoxA from B. infantis ATCC 15697 in the O 2 -tolerant strain B. minimum DSM 20102 resulted in growth inhibition at O 2 concentrations above 10% (28). B. minimum DSM 20102 has not been included in the homology search of our study due to its incomplete genome sequence at NCBI, but a separate online BLASTp search, using default settings, revealed that B. minimum DSM 20102 possesses an NPOX homolog itself, which most likely is not expressed at high levels under aerobic conditions. The lowest sequence similarity (,60%) was detected for NPOX homologs in strains of the B. pseudolongum and B. asteroides group. Multiple-sequence alignment of all identified NPOX hits revealed that the homologs in B. animalis possess a unique motif (P-xxx-S/T-xxxxx-C, positions 59 to 71) that is similar to the active-site motif of peroxiredoxins (75). Thus, the activity of the NPOX homologs in these species might vary from that in other species.
Only B. longum subsp. longum NCC2705, described to possess relatively high intrinsic H 2 O 2 tolerance (58), and a few other B. longum and B. bifidum strains were found to lack an NPOX homolog (Fig. S1). It is possible that the absence of the H 2 O 2 -forming activity of NPOX reduces the burden on the strain's H 2 O 2 -detoxifying enzymes from intracellularly formed H 2 O 2 and free capacity for the detoxification of extracellular H 2 O 2 . Therefore, it should be assessed if the strains lacking a homolog of NPOX show higher O 2 tolerance than other strains of the species. Another enzyme that might contribute to H 2 O 2 formation is the L-aspartate oxidase, which catalyzes the first step of de novo NAD biosynthesis by oxidizing aspartate to iminoaspartate. It has been suggested that both fumarate and O 2 can serve as electron acceptors for L-aspartate oxidase, while using the latter results in the formation of H 2 O 2 (76). Even though a previous study showed that fumarate is the preferred in vivo substrate of L-aspartate oxidase in Escherichia coli, H 2 O 2 formation was detected under aerobic conditions when fumarate levels were low (76). Thus, the activity of L-aspartate oxidase might contribute to O 2 sensitivity by facilitating H 2 O 2 formation. In line with this conclusion, our results indicated that homologs of L-aspartate oxidase are present primarily in species known to be rather O 2 -sensitive, whereas relatively O 2 -tolerant species, such as B. animalis, lacked an L-aspartate oxidase homolog (Fig. 1).
(v) The absence of multiple ROS detoxification enzymes may cause hypersensitivity to oxygen. Until now, no H 2 O-forming NADH oxidase (NOX) has been purified and characterized from a Bifidobacterium strain. However, an NADH oxidase upregulated in the O 2 -tolerant B. animalis subsp. lactis IPLA4549 upon oxidative stress was suggested to contribute to detoxification of O 2 to H 2 O (20). Since the genome sequence of B. animalis subsp. lactis IPLA4549 is not publicly available, NOX from B. longum subsp. longum NCC2705 was used as a query gene. Based on our genomic analysis, all strains of 73% of the studied species lacked a homolog of the putative H 2 O-forming NADH oxidase (Fig. 1). A previous study on the H 2 O-NADH oxidase of Enterococcus faecalis 10C1 revealed that a single cysteine (Cys42) in the active site is determinative of the fourelectron reduction of O 2 to H 2 O in the NADH oxidase (77). Multiple-sequence alignment of the bifidobacterial NOX and the NOX of E. faecalis 10C1 confirmed the presence of the active-site cysteine in all analyzed genes (Cys43/44) and showed similarity in the surrounding amino acid sequence. Thus, the NADH oxidase of all Bifidobacterium strains would be expected to have H 2 O-forming activity instead of H 2 O 2 -forming activity, as was previously suggested for B. longum subsp. longum NCC2705 (27). Overall, species that lacked a NOX homolog were highly diverse in terms of O 2 -sensitivity, including strains of the O 2 -hypersensitive species B. adolescentis and B. angulatum but also isolates from the digestive tract of bees. Therefore, the absence of a NOX homolog does not seem to be determinative of the O 2 sensitivity of strains.
Several studies found a positive correlation between O 2 tolerance of Bifidobacterium strains and the ability to detoxify H 2 O 2 (20,69,70). Genomics and proteomics have revealed that bifidobacteria lack a gene for NADH peroxidase (35,78) and that an alkyl hydroperoxide reductase subunit C (AhpC) and a thioredoxin reductase-like protein (TrxR), encoded next to AhpC, instead may be responsible for H 2 O 2 decomposition (25,27,35,78). Our results showed that in 78% of the analyzed species, all strains harbored both an AhpC and TrxR homolog (Fig. 1). No strain possessed only one of the genes, which reaffirms the assumption of their coactivity (Fig. S1). The presence of the AhpC-TrxR system in O 2 -sensitive strains, such as B. longum subsp. infantis ATCC 15697, suggests that in these strains the H 2 O 2 detoxification activity provided by the system is insufficient to cope with H 2 O 2 formation under aerobic conditions. Strains of the species B. angulatum, including B. angulatum DSM 20098, reported to be highly O 2 -sensitive (Table S3), B. dentium, B. lemurum, B. subtile, and some strains of B. adolescentis, including B. adolescentis ATCC 15703, which is also known to be highly O 2 sensitive (Table S3), lacked both genes ( Fig. 1 and Fig. S1). A second gene encoding a thioredoxin reductase (TrxR), which has been identified in B. longum subsp. longum NCC2705 and B. bifidum JCM1255, that lacks the N-terminal domain of the disulfide reductase AhpF and seems not to be induced by O 2 stress (13, 25) was found to be highly conserved across genomes of species included in this study (Fig. 1).
Besides the AhpC-TrxR system, the bacterioferritin comigratory protein (BCP), first detected in B. longum subsp. longum NCC2705 (described as thioredoxin-dependent thiol peroxidase) (35) and induced in relatively H 2 O 2 -tolerant B. animalis subsp. lactis 01 upon oxidative stress (16), may contribute to thioredoxin-dependent H 2 O 2 -peroxidase activity in Bifidobacterium strains. In E. coli and Helicobacter pylori, BCP shows a substrate preference for linoleic acid hydroperoxide over H 2 O 2 (79, 80), but the substrate specificity of BCP in bifidobacteria remains to be investigated. Homologs of BCP from B. longum subsp. longum NCC2705 were found in all strains of 55% of the studied species (Fig. 1). The analysis conducted in our study showed an overlap in the subset of strains that lacked the AhpC-TrxR system and strains that lacked a BCP, including all B. angulatum and B. dentium strains (Fig. 1) and some B. adolescentis strains, including B. adolescentis ATCC 15703 (Fig. S1). In addition, species of the B. asteroides group and B. subtile lacked a BCP homolog (Fig. 1).
Since BCP from B. animalis and B. pseudolongum strains shows low sequence identity to the query gene (Fig. 1), the sequence motifs of the BCP gene products were further compared in a multiple-sequence alignment. In general, the active-site motif of peroxiredoxins (P-xxx-T/S-xx-C) includes a conserved cysteine (peroxidatic cysteine) that acts with the peroxide (R-O-O-R) to form a cysteine sulfenic acid (R-SOH) (75). Some peroxiredoxins contain a second cysteine (resolving) that can form a disulfide bond with R-SOH under the formation of H 2 O that is later reduced by a thiol-containing disulfide reductase system (75). In contrast, R-SOH in peroxiredoxins lacking a second cysteine form an intermolecular disulfide bond with a thiol of another protein or small molecule (75). The multiple-sequence alignment revealed that BCPs from B. animalis lacked a resolving cysteine in the active site, while the BCPs of all other species possess a second cysteine and share a motif (P-xxx-T/S-xx-C-xxxx-C) (Fig. 4). Besides the absence of the resolving cysteine in strains of three species, the motif around the catalytic cysteine in BCP showed additional variations across species (Fig. 4).
Additional ROS detoxification enzymes detected in bifidobacteria included the DNA-binding protein from starved cells (Dps) and the peptide-methionine sulfoxide reductase (MsrA/B). Dps protects cells from oxidative damage through binding genomic DNA and by sequestering free Fe 21 and H 2 O 2 that could otherwise react in Fenton reactions, resulting in OH Á formation (81). MsrAB reduces methionine sulfoxide (resulting from the oxidation of methionine) back to methionine and thereby scavenges ROS (82). Strains of 27% of the studied species were found to lack a homolog of Dps (Fig.  1), including highly O 2 -sensitive strains of B. adolescentis and B. angulatum as well as relatively O 2 -tolerant strains of B. thermophilum (Table S3). In contrast, multiple B. longum and B. breve strains were found to possess a second copy of Dps.
All Dps-negative strains also lacked a gene encoding MsrAB (Fig. S1). MsrAB was further absent from nine additional strains of B. dentium, B. choerinum, and B. pullorum subsp. gallinarum and isolates from lemurs. Due to the various O 2 sensitivities of strains lacking a Dps and MsrAB homolog, their sole absence appears not to be indicative of O 2 sensitivity.
Overall, studying the prevalence of genes associated with the oxidative stress response in bifidobacterial genomes showed that the absence of single stress-associated gene products, such as Dps, AhpC, TrxR, and MsrAB, may not be decisive in the sensitivity of a strain to O 2 . Instead, the results indicated that the lack of multiple ROS-detoxifying genes, as observed for B. angulatum and some B. adolescentis strains, might cause exceptionally high sensitivity to O 2 . Strains of B. dentium, which have not been described as being particularly O 2 -sensitive, lacked the same ROS-detoxifying enzymes as B. angulatum and some B. adolescentis strains except for Dps ( Fig. 1 and Fig. S1). Comparing the O 2 tolerance of B. dentium with B. angulatum and B. adolescentis might provide some insights into the importance of Dps for survival under aerobic conditions. Moreover, comparing the O 2 tolerance of B. adolescentis strains that were found to harbor AhpC, TrxR, and BCP with those that

Stress-Associated Genes in Bifidobacteria
Applied and Environmental Microbiology lacked these enzymes could validate the significance of these three ROS-detoxifying enzymes for O 2 tolerance. Surprisingly, O 2 -tolerant strains of the species B. animalis subsp. lactis harbored almost the same set of O 2 -scavenging and ROS-detoxifying enzymes as O 2 -sensitive strains of B. longum, B. breve, and B. bifidum (Fig. S1). Gene regulation, the ratio of different enzymes involved in the ROS-detoxification system (25), as well as the efficiency of NAD(P)H production driving the oxidative stress response may vary between these Bifidobacterium species. More knowledge on the functions of individual enzymes as well as on O 2 tolerance of individual strains will need to be collected to allow further connections between genotypes and O 2 sensitivity.
Acid stress response. Based on the upregulation and increased activity of the F 1 F 0 -ATP synthase upon acidic stress, the enzyme has been suggested to improve pH homeostasis by ATP-driven extrusion of protons (10,11,64,83). As stated previously, the genes encoding F 1 F 0 -ATP synthase homologs were highly conserved across the analyzed species (Fig. 1), but its primary role might be different in putative respiring and nonrespiring strains.
The prevalence of nine additional genes associated with acid stress among bifidobacterial genomes was assessed in this study. Three of the genes, namely, polyphosphate kinase (PPK1), glutamate-cysteine ligase (GCL), and aminopeptidase PepP, were found to be highly conserved (Fig. 1). PPK1 is responsible for the formation of polyphosphate granules, which was found to contribute to acid stress resistance in B. scardovii JCM 12489 (14). Polyphosphate granules may promote stress resistance by serving as a reserve energy source and contributing to pH homeostasis (84). GCL and PepP were reported to be upregulated upon acid stress in B. longum strains (10,11,83). GCL catalyzes the formation of the glutathione precursor g-glutamylcysteine. Since bifidobacteria lack a candidate gene encoding glutathione synthase (based on functional annotation information in NCBI GenBank), it has been suggested that g-glutamylcysteine itself has a protective function under acid stress (11). The high degree of conservation of PepP across Bifidobacterium species, which may supply free amino acids for synthesis and repair of proteins under acidic conditions from peptides, agrees with the high degree of conservation observed for other members of the PQC system.
Oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase are responsible for the detoxification of oxalate, a strong acid present in plant-based food. The expression of both enzymes was found to be induced upon acid stress in the acid-tolerant B. dentium Bd1 (12). Our genomic analysis showed that oxalyl-CoA decarboxylase and formyl-CoA transferase homologs from B. dentium Bd1 were only present in strains from the three species in the B. pseudolongum group, in B. pseudocatenulatum strains, and in other B. dentium strains (Fig. 1). Strains of these species have previously been shown to be acid tolerant (Table S3). The detoxification of oxalate, which is further linked to the scavenging of a proton (85), may contribute to coping with acidic conditions. However, the presence of oxalyl-CoA decarboxylase and formyl-CoA transferase homologs can only explain their acid tolerance in the presence of oxalate.
Enzymes involved in amino acid metabolism in Bifidobacterium strains were proposed to contribute to pH homeostasis upon acid stress due to proton scavenging, including the glutamate decarboxylase (GadB) pathway. GadB catalyzes the conversion of glutamate to gamma-aminobutyrate (GABA), consuming an intracellular proton, while a glutamate/gamma-aminobutyrate antiporter (GadC) transports GABA out of the cell and exchanges it with another glutamate (86). Genes encoding GadC and GadB were found to be upregulated in B. dentium Bd1 when subjected to acid stress (12). Homologs of the two gene products from B. dentium Bd1 were only found in B. adolescentis, B. angulatum, and other B. dentium strains (Fig. 1). The ability to produce GABA and the prevalence of gadB and gadC genes in their genome also was described for B. moukalabense, B. stercoris, B. merycicum, and B. ruminantium, which are not included in this study (87). A B. longum subsp. infantis strain was suggested to be a GABA producer (88), but none of the 11 B. longum subsp. infantis strains examined here were found to have GadC or GadB homologs (Fig. S1).
In the acid-resistant water kefir isolates B. aquikefiri CCUG 67145 T and B. tibiigranuli TMW 2.2057 T (89), a glutamine-ABC transporter, asparagine synthetase, asparaginase, and asparagine permease have been suggested to form a pathway that contributes to pH homeostasis under acid conditions by ammonia formation, which can scavenge protons in the cytoplasm (18). Like the genes encoding the glutamate decarboxylase pathway, genes encoding asparaginase and asparagine synthetase were only found in B. adolescentis, B. angulatum, and B. dentium (Fig. 1), whereas a few strains of other species possess one of the two genes (Fig. S1). In contrast to strains of B. dentium, strains of B. adolescentis and B. angulatum are not known to be particularly acid tolerant (Table S3) (34,61). Thus, assessing the presence of the genes of the glutamate decarboxylase pathway and the described asparagine synthetase/asparaginase pathway appears to be insufficient to infer acid tolerance.
Bile stress response. Bile salt hydrolases (BSHs), which catalyze the deconjugation of primary and secondary bile salts, have been extensively studied in the context of bile salt resistance of bifidobacteria. However, it remains unclear if and how the activity of BSHs contributes to higher bile stress resistance (90,91). Several BSHs have been characterized in bifidobacteria (92)(93)(94)(95), where they exhibit low sequence similarity among different species (96). BSH homologs were found in 94% of the strains analyzed in this study and showed high sequence variation (Fig. 1). The lowest sequence identity was detected in strains of B. angulatum (47%). In agreement with their natural environment, no BSH homolog was detected in isolates from the digestive tract of bees or isolates from lemurs. Interestingly, lemurs lack the ability to produce glycine-conjugated bile salts (97), which are commonly the preferred substrate of bifidobacterial BSH (93,94,98) and are associated with higher toxicity than the taurine-conjugated bile salts (99,100). This suggests that the absence of a BSH-encoding gene in the genomes of isolates from lemurs is a result of habitat adaptation or that these strains colonize lemurs, as they cannot survive in other animals that produce glycine-conjugated bile salts. Moreover, no BSH homologs were detected in B. pullorum subsp. gallinarum CACC 514 and B. scardovi JCM 12489 (only representative of B. scardovi species) (Fig.  S1); however, the biological significance of this remains unknown.
Organic solvent stress response. Little is known about the stress response in bifidobacteria to organic solvents. However, an oleate hydratase in B. breve NCFB 2258 has previously been suggested to contribute to tolerance to butanol (2). Homologs of the oleate hydratase from B. breve NCFB 2258 were detected in strains of most species (Fig. 1). Strains lacking homologs were strains of the B. asteroides group, B. subtile, B. eulemuris DSM 100216 (only representative of B. eulemuris species), B. longum BORI and CCUG 30698, and B. animalis subsp. lactis ATCC 27673, which has already been described as having a genetic content distinct from that of other strains of the species (101).
Putative transcriptional regulators of stress response. In addition to the regulatory mechanism described for the PQC and DNA repair systems, the presence of homologs of additional stress-associated transcriptional regulators identified in Bifidobacterium strains was investigated (9,13).
All strains analyzed in this study possessed the housekeeping sigma factor HrdB (homolog of RpoD) (Fig. 1). Moreover, all strains, except for B. pullorum subsp. gallinarum CACC 514, B. coryneforme LMG18911, and B. indicum LMG 11587, also possessed a homolog of an alternative sigma factor, extracytoplasmic function (ECF) RNA polymerase sigma factor RpoE, in their genomes (RpoE_1 in Fig. 1). In some strains included in the study, the RpoE was shorter (211 to 227 amino acids) than the query gene product from B. breve UCC2003 (253 amino acids), resulting in a query coverage between 71% and 79% (Data Set S1). Alternative sigma factors are known to control specialized regulons, e.g., regulons of stress response genes (102). However, very little is known about the function of RpoE in the stress response in bifidobacteria. The presence of RpoE has previously only been described in the genomes of B. breve UCC2003 and B. longum strains (13, 47, 50, 103), and which genes are controlled by RpoE still needs to be investigated, e.g., by applying DNase footprinting. Due to the absence of a consensus binding motif in the promoter region of the hsp20 gene, it has been suggested that RpoE regulates the expression of Hsp20 (30). However, this hypothesis is contradicted by the observation that a negative effector of RpoE is upregulated in B. longum subsp. longum NCC2705 upon heat stress and the expression of hsp20 is induced simultaneously (13). Moreover, whereas RpoE was found to be widely conserved in our analysis, Hsp20 homologs were only found in a subset of species, mainly representing isolates from humans (Fig. 1).
A second copy of RpoE was identified in 36 strains from B. breve and B. longum and in isolates from bees, including B. asteroides, B. coryneforme, and B. indicum (RpoE_2 in Fig. 1). Interestingly, all strains possessing a second copy of RpoE were also found to harbor a gene encoding a homolog of the putative SOD from B. longum subsp. infantis ATCC 15697. In B. longum subsp. infantis, the gene encoding RpoE (Blon_1402) is close to the gene encoding the putative SOD (Blon_1406), so it is conceivable that RpoE plays a role in transcriptional regulation of the putative SOD. In general, the presence of RpoE in all strains and a second copy in some strains indicates an important role of the sigma factor in transcriptional regulation.
In a previous study, two general types of WhiB-type regulators, WhiB2 and WblE, were detected in Bifidobacterium strains (9). WhiB-like proteins are thought to function as transcriptional regulators of major cellular processes, including stress responses, in Actinobacteria (104). While wblE orthologs have been suggested to be highly conserved, no whiB2 orthologs have been identified in B. animalis subsp. lactis and B. gallicum strains (9). In addition, some strains have been found to have additional whiB-like genes with various lengths and low sequence similarity and were suggested to provide an additional benefit for sensing external signals (9). In B. longum B379M, the expression of wblE was induced upon exposure to various stressors, whereas no stress-induced transcription was observed for whiB2 (9). In contrast, B. breve UCC2003 showed upregulation of the whiB2 gene upon osmotic stress (see supplemental material in reference 24).
The analysis conducted in our study verified the presence of a WblE homolog in all strains of the analyzed species and revealed a high conservation in terms of its protein sequence (minimum of 88% identity). Moreover, the results confirmed that strains of B. animalis lack a WhiB2 homolog (Fig. 1). In addition, no WhiB2 homologs could be detected in genomes of other animal isolates (Fig. 1). Homologs of a second WhiB-like protein from B. longum subsp. longum NCC2705 (9, 35) were found in genomes of some strains of the B. longum group, B. catenulatum subsp. kashiwanohense and B. kashinowehense (Whib-like protein_1 in Fig. S1). Homologs with very low identity (43% to 50%) were detected in B. subtile and B. pullorum subsp. gallinarum (Fig. S1), suggesting that these WhiB-like proteins differ in function compared to the protein in B. longum subsp. longum NCC2705. Homologs of two additional WhiB-like proteins of the well-characterized bee isolate B. asteroides PRL2011 across strains of this study were assessed (Whiblike protein_2 and Whib-like protein_3). Whereas high-identity homologs were found for one of the genes in strains of various phylogenetic groups, the second appeared to be present only in B. asteroides and B. thermophilum ( Fig. 1 and Fig. S1). Overall, the role of WhiB-like proteins in stress responses remains to be further investigated; however, the diverse prevalence of WhiB-like proteins in bifidobacteria suggests that their role in the transcriptional regulation of genes has variable impacts among strains and species.
Conclusions. Our analysis revealed multiple differences in the stress-associated genetic makeup of different Bifidobacterium species as well as some variations among strains of the same species, e.g., B. adolescentis. While genes encoding molecular players of the PQC and DNA repair systems were found to be highly conserved across species, the regulatory mechanisms of these systems might differ between phylogenetic groups. The presence/absence pattern of genes linked to oxidative stress response could not fully explain variations in the O 2 tolerance across species, suggesting that their expression level upon oxidative stress influences O 2 sensitivity. However, the absence of several ROS-detoxifying enzymes in strains of B. angulatum and B. adolescentis most likely contributes to their O 2 sensitivity. Species-dependent genes associated with acid stress response might be a result of adaptation to natural habitats. Similarly, the presence of a gene encoding a bile salt hydrolase in human and mammalian isolates appeared highly linked to the presence of bile salts in their natural habitat. Moreover, the distribution of putative regulators of various stress responses varied across species, indicating that the involvement of different sets of regulators contributes to the highly diverse stress physiology of Bifidobacterium species.
In conclusion, mining bifidobacterial genomes for homologs of genes associated with various stress responses and their regulatory elements provided new insights into the molecular mechanisms underlying their diverse stress physiology. The obtained results lay out the foundation for hypothesis generation in future studies. However, systematic assessment of the effect of various stressors on growth and survival of sequenced Bifidobacterium strains as well as the identification of the biological function of stress-induced genes is required to obtain stronger correlations between the presence of stress-associated genes and stress tolerance of different strains.

MATERIALS AND METHODS
Identification of representative stress-associated genes. Previous studies on stress response in bifidobacteria were consulted to identify representative genes that have been implicated in the stress response of bifidobacteria (see Table S1 in the supplemental material) (2-32, 35, 59, 64, 65). For the genomic analysis, the protein sequence of each representative gene was extracted from the genome sequence of a Bifidobacterium strain in which it was proposed to be involved in stress responses. Moreover, NCBI GenBank was searched for bifidobacterial genes annotated as superoxide dismutase and L-aspartate oxidase.
Selection of strains. All publicly available complete genome sequences of Bifidobacterium strains were retrieved from the NCBI RefSeq database, resulting in 171 genomes representing 22 out of 54 currently recognized Bifidobacterium species (Table 1) (61). All strains included in the study are listed in Table  S2. Including only complete RefSeq genomes was intended to minimize the effect of fragmented genes in incomplete draft genomes, which might be missed with strict homology search parameters. The genome sequences were retrieved using the ncbi-genome-download package (K. Blin, https://github.com/kblin/ncbi -genome-download) with the arguments "-genera Bifidobacterium -section refseq -assembly-levels complete." The RefSeq genomes include structural and functional annotations computed through NCBI's PGAP.
Homology search. The proteomes of the 171 Bifidobacterium strains were searched for homologs of the representative stress-associated gene products using the DIAMOND BLASTp algorithm (v2.0.9.147) (105). For each genome, only the hit with the highest score for a given query was selected. However, information on the presence of additional hits was also collected. If a gene was the best hit for multiple query genes, only the best hit was retrieved. Default settings were used for most parameters, including E value (0.001), while applying a cutoff of 40% sequence identity across 70% of the protein sequence length. The maximum number of returned hits was set to 1,000 (default, 25). The results of the homology search were summarized by identifying the sequence identity of the highest hit for each query sequence in each bifidobacterial genome. Since there was only very limited variation in the presence of most stressassociated genes across strains of the same species (Fig. S1), the results were aggregated by species. This was done by calculating the median sequence identity of the best hit for a given query across all strains of each species. To visualize the presence of stress-associated genes in an evolutionary context, the phylogeny of the strains included in the study was constructed using the housekeeping gene dnaA. A maximum likelihood phylogeny of the dnaA sequences was constructed in CLC Genomics Workbench 20.0 with default settings, using the dnaA sequence from Gardnerella vaginalis (WP_004114028.1) as an outgroup. Bifidobacterium species were assigned to the previously suggested phylogenetic groups, including B. adolescentis, B. asteroides, B. boum, B. longum, B. pseudolongum, and B. pullorum, except for the strains of six species that have not yet been unambiguously assigned to any phylogenetic group (37,40).
Genome-wide in silico screening for known binding motifs. The genomes of B. animalis subsp. lactis BB-12, B. adolescentis ATCC 15703, and B. longum subsp. longum NCC2705 were screened for the presence of previously proposed consensus sequences of the three transcriptional regulators HspR, HrcA, and ClgR (24). The online version of FIMO was used for the analysis (53). The threshold for the P value (probability of a random sequence of the same length as the motif matching the position of the sequence with as good or better score) was set to 0.0001, and the q-value (false discovery rate if the occurrence is accepted as significant) was set to 0.01 (53).
Multiple-sequence alignments. Multiple-sequence alignments were run in CLC Genomics Workbench 20.0 with default settings, using the progressive alignment mode "very accurate (slow)." Search for HemW-specific motifs in HemN homologs of Bifidobacterium strains. The protein sequences of HemN in B. longum subsp. longum NCC2705, B. animalis subsp. lactis BB-12, and B. asteroides PRL2011 were compared to the protein sequence of HemW in Lactococcus lactis through multiplesequence alignment and were subsequently screened for conserved amino acid residues specific for HemW-like proteins, including (i) His 134 , (ii) H 184 VxxYxLxLE, (iii) Y 234 ExS, and (iv) H 248 NxxYW (72).
Data availability. We declare that all the data supporting the work are available within the paper and its supplemental material.